B. Frølund

Extraction of extracellular polymers from activated sludge using a cation exchange resin

Abstract The extraction of water soluble extracellular polymeric substances (EPS) from activated sludge was investigated. The extraction procedure was based upon cation exchange using a cation exchange resin (CER). Activated sludge from two different types of treatment plants responded very similarly to the extraction procedure. The EPS yield was enhanced by increasing the stirring intensity, the amounts of CER added and by increasing the extraction time. For the chosen extraction procedure the yield was twice as high as other commonly used procedures. The extract consisted mainly of protein but also humic compounds, carbohydrates, uronic acids and DNA were found in significant amounts. The extracted amounts and relative fraction of the individual compounds strongly depended on how the extraction was performed. The ratio between protein and carbohydrate was found in the range 3.9–5.1 depending on the extraction time. Humic compounds and DNA were the compounds most easily extracted. HPSEC investigation of the extract revealed that the extraction did not significantly degrade the EPS. Some cell lysis was identified during the extraction for extraction times greater than 1–2 h by observing a decrease in cell number (stained by DAPI, CTC and acridine orange). The lysis was not considered a significant problem for contaminating the EPS. Measurements of the cell number and cell size distribution in the sludge suggested that the cell mass did not account for more than approximately 10–15% of the total organic fraction of the investigated sludge. Two extraction strategies were formulated. One for a very gentle and one for a very effective method. Analytical methods for analysis of sludge and EPS extracts were compared and discussed. A corrected Lowry method for analyzing protein as well as humic compounds was implemented and found suitable.

Enzymatic activity in the activated-sludge floc matrix

The enzymatic activity of activated sludge was investigated with special emphasis on the localization of the enzymes in the sludge floc matrix. Activated sludge from an advanced activated-sludge treatment plant, performing biological N and P removal, was used. An enzymatic fingerprint was established using a panel of six different enzymes. The fingerprint revealed peptidase as the most dominating specific enzyme tested. By monitoring sludge bulk enzymatic activity over a 3-month period using fluorescein diacetate as an enzyme substrate, considerable variations in activity were observed even over short periods (a few days). The variation in esterase activity was to some extent correlated to the presence of humic compounds in the sludge, but not to the sludge protein content. Comparison of full sludge enzyme activity to the activity of a batch-grown sludge culture indicated that enzymes accumulated in sludge flocs. A large proportion of the exoenzymes were immobilized in the sludge by adsorption in the extracellular polymeric substances (EPS) matrix. This was demonstrated by extraction of EPS from the activated sludge using cation exchange. Contemporary to the release of EPS a very large fraction of the exoenzymes was released into the water. This showed that the exoenzymes should be considered to be an integrated part of the EPS matrix rather than as direct indicators of the microbial activity or biomass.

Changes in the composition of extracellular polymeric substances in activated sludge during anaerobic storage.

Changes in the chemical composition of organic compounds in total activated sludge, activated sludge extracellular polymeric substances (EPS), and sludge bulk water during anaerobic storage (12 days) were studied. The background for the study was that anaerobic storage of activated sludge, which often takes place at wastewater treatment plants before dewatering, causes a deterioration of the dewaterability. The reasons are not known at present, but may be related to changes in exopolymer composition of the flocs. The results showed that a fast decrease in total sludge protein and carbohydrate took place within 3 days of anaerobic storage as a result of degradation processes, which accounted for approximately 20% of the organic fraction. The amount of uronic acids and humic compounds remained almost constant in the sludge. The EPS were extracted from the floc matrix using a cationexchange resin. In the EPS matrix a similar initial (2–3 days) degradation of proteins and carbohydrate took place, whereas the content of DNA and uronic acids showed minor changes. The extractability of humic compounds increased during the first 3 days of storage. No changes in extractability of the carbohydrate were observed. A fraction of the EPS protein was found to be difficult to extract but was observed to be degraded during the anaerobic storage. The EPS composition was further characterized by high-performance size-exclusion chromatography analysis obtained by on-line UV detection and post-column detection of proteins, carbohydrates, humic compounds and DNA. Four fractions of polysaccharides were found, of which only one was responsible for the decrease in the carbohydrate content observed with storage time. The fraction was presumably of low molecular mass. Humic compounds and volatile fatty acids (acetate and propionate) were released to the bulk water from the flocs during the storage. A possible mechanism to explain the reduced dewaterability developed during anaerobic storage, partly because of the observed changes in EPS, is discussed.

Monitoring of microbial souring in chemically treated, produced-water biofilm systems using molecular techniques.

The identification of bacteria in oil production facilities has previously been based on culture techniques. However, cultivation of bacteria from these often-extreme environments can lead to errors in identifying the microbial community members. In this study, molecular techniques including fluorescence in situ hybridization, PCR, denaturing gradient gel electrophoresis, and sequencing were used to track changes in bacterial biofilm populations treated with nitrate, nitrite, or nitrate + molybdate as agents for the control of sulfide production. Results indicated that nitrite and nitrate + molybdate reduced sulfide production, while nitrate alone had no effect on sulfide generation. No long-term effect on sulfide production was observed. Initial sulfate-reducing bacterial numbers were not influenced by the chemical treatments, although a significant increase in sulfate-reducing bacteria was observed after termination of the treatments. Molecular analysis showed a diverse bacterial population, but no major shifts in the population due to treatment effects were observed.

Implementation of an HPLC polystyrene divinylbenzene column for separation of activated sludge exopolymers

A high-pressure size-exclusion chromatography procedure for separation of activated sludge exopolymers was investigated and implemented in order to achieve a documented and faster separation procedure than the conventional low-pressure size-exclusion chromatography methods previously suggested in studies of activated sludges from a traditional and an advanced from activated sludges from a traditional and an advanced activated sludge treatment plant performing biological nitrogen and phosphorus removal were used. For both types of exopolymers the separation was largely dependent on the mobile-phase. Using NaCl and ortho-phosphate in the molar proportion 10:1 it was shown that for a mobile-phase ionic strength of 0.011 and pH in the range 7.0–10.0 no irreversible column adsorption occurred. For a standard procedure a mobile-phase pH of 7.0 was selected in order to separate the exopolymers into the maximal number of peaks. Alterations in the mobile-phase, i.e. using a pH below 7.0 or a mobile-phase ionic strength above 0.011, changed the separation for both types of exopolymers and caused irreversible column adsorption. Similarly, using deionized water as the mobile-phase irreversible column adsorption was introduced and the separation was strongly affected. The method applicability for qualitative characterization of exopolymers was demonstrated. The method was found to be successful in showing differences and similarities between exopolymers from two different activated sludge treatment plants, showing degradation of exopolymer compounds due to exoenzymes in the exopolymers and showing that snow melting and subsequent high conductivity in the inlet to the waste-water treatment plant had an impact on the chromatographic fingerprint of the extracted exopolymers.

Microbial diversity in biofilms from corroding heating systems

Culture-independent investigations of the bacterial diversity and activity in district heating systems with and without corrosion did not make it possible to relate one group of microorganisms with the observed corrosion. Fluorescence in situ hybridization by oligonucleotide probes revealed the dominance of β-proteobacteria, sulphate reducing prokaryotes and α-proteobacteria. Analysis of a clone library from one Danish heating (DH) system showed that the most sequences formed two clusters within the α-proteobacteria affiliated to the families Rhizobiaceae and Acetobacteraceae and two clusters within the β-proteobacteria belonging to the family Comamonadaceae. Functional groups were determined by microautoradiography showing aerobic and anaerobic bacteria (sulphate reducing and methanogenic bacteria). The corrosion study showed that pitting corrosion rates were five to ten times higher than the general corrosion rates, suggesting the presence of biocorrosion. The results indicate that several bacterial groups could be involved in corrosion of DH system piping including sulphate reducing prokaryotes, Acidovorax (within the β-proteobacteria), methanogenic bacteria and others.

Microbial Fe(III) Reduction in Activated Sludge

Summary This study examined the concentration of iron, the rate of Fe(III) reduction and the numbers of Fe(III)-reducing bacteria present in activated sludge from 10 different wastewater treatment plants throughout Denmark. The temporal variation in Fe(III) reduction rates and the effect of temperature and various electron donors on the Fe(III) reduction rate in activated sludge within a selected treatment plant in Aalborg, Denmark were also examined. A Fe(III)-reducing bacterium that can grow using the Fe(III) bound within activated sludge as a sole electron acceptor was isolated from the sludge. These studies demonstrate that Fe(III)-reducing bacteria are present in the activated sludge of Danish wastewater treatment plants as normal members of the microbial community and that Fe(III) is a significant electron acceptor in this environment.

The effect of alkaline pH conditions on a sulphate reducing consortium from a Danish district heating plant

This study demonstrated that a non‐alkaliphilic sulphate reducing bacterial (SRB) consortium could survive the alkaline environment in the circulation water of a Danish District Heating Plant. The plant was experiencing corrosion, and SRB were postulated to be responsible. A bacterial consortium containing mesophilic SRB was enriched in vials inoculated with the plant circulation water. A batch experiment illustrated the ability of the SRB to survive a pH of 10.5. The same SRB produced sulphide at a pH of 9.3, when the pH in the batch vials was lowered by other bacteria present in the consortium. Another experiment demonstrated that, when protected in a biofilm, the same SRB consortium produced sulphide at a bulk fluid pH of 10.2. These results could be implemented as part of a SRB control strategy in the field.

Biocorrosion and biofilm formation in a nutrient limited heating system subjected to alternating microaerophilic conditions

Severe biofilm formation and biocorrosion have been observed in heating systems even when the water quality complied with existing standards. The coupling between water chemistry, biofilm formation, species composition, and biocorrosion in a heating system was investigated by adding low concentrations of nutrients and oxygen under continuous and alternating dosing regimes. Molecular analysis of 16S rRNA gene fragments demonstrated that the amendments did not cause changes in the overall bacterial community composition. The combined alternating dosing of nutrients and oxygen caused increased rates of pitting (bio-) corrosion. Detection of bacteria involved in sulfide production and oxidation by retrieval of the functional dsrAB and apsA genes revealed the presence of Gram-positive sulfate- and sulfite-reducers and an unknown sulfur-oxidizer. Therefore, to control biocorrosion, sources of oxygen and nutrients must be limited, since the effect of the alternating operational conditions apparently is more important than the presence of potentially corrosive biofilm bacteria.